Abstract: Ubiquitination is a highly conserved and essential post-translational modification that controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by E3 ubiquitin ligases which are responsible for substrate specificity. E3 ligases catalyze the formation of an isopeptide bond between a lysine residue (or the N-terminus) of the substrate and ubiquitin. Plasma membrane proteins that are ubiquitinated are subject to endocytosis and degradation in the lysosome. The details of how ubiquitination regulates internalization and targeting to the lysosome remain unclear in higher order eukaryotes. The Kaposi's Sarcoma-associated Herpesvirus encodes two homologous E3 ligases, MIR1 and MIR2 that mediate the ubiquitination of several cell surface proteins, thus modulating the protein composition of the plasma membrane of infected cells. Among the proteins removed from the cell surface are the MHC-1 molecules which are critical in the recognition of virally infected cells by the immune system. Therefore, understanding the mechanism by which the MIR proteins ubiquitinate substrates and target them for degradation is essential to understanding viral pathogenesis and the process of ubiquitination. Here we found that the MIR proteins and some of their homologs have the unique ability to downregulate surface expression of their substrate by mediating ubiquitination on cysteine residues. Characterization of this novel form of ubiquitination reveals that it shares many similarities with the conventional ubiquitination of lysine residues. In particular, the location of the lysine or cysteine residue within the intracytoplasmic tail of the substrate is critical for efficient ubiquitination. The positions that are most well-suited for cysteine-mediated downregulation are very similar to the positions favorable for lysine-mediated downregulation. Furthermore, we do not find any evidence of a cofactor that is specifically necessary for cysteine-ubiquitination. Our results suggest that lysine- and cysteine-ubiquitination are regulated in a common manner and may involve a similar mechanism.